Invasive fungal infections are still the cause of major complications in immunocompromised patients (patients receiving immunosuppressive therapy—such as bone marrow and organ transplant recipients, cancer patients, and HIV/AIDS patients). Due to the emergence of pathogens resistant to conventional antifungals agents and toxicity of some antimycotics, intense efforts are being made in antifungal drug discovery to develop more promising and effective antifungal agents for clinical use.
Many currently available and clinically used antimicrobial drugs have undesirable toxic and other side effects. In addition, a wide-spread use of these drugs has lead to the rapid development of drug-resistant strains which are the main cause for the treatment failures. Thus, development and delivery of new antimicrobial agents with different mechanism of action, low toxicity and low tendency to elicit resistance is urgently needed. Among other approaches, naturally occurring cationic antimicrobial peptides is attracting increasing attention. This is because unlike many currently used antimicrobial compounds (CAMPs), they show little or no toxicity toward mammalian cells and low tendency to elicit resistance.
We have previously shown that peptides derived from the N-terminal region of the low molecular mass human salivary mucin, MUC7, have a significant and a broadspectrum fungicidal and bactericidal activity in vitro, as determined by killing assays in phosphate buffer (1-3). A further study showed that MUC7 12-mer (amino acids 40-51 of the parent MUC7, with net charge of +6) is the optimal size peptide fragment that possesses potent antifungal activity against Candida albicans and Cryptococcus neoformans (4). A clear correlation between the net positive charge of the MUC7 12-mer, its potency and initial interaction of peptide with fungal cells was found by killing assays, fluorescence microscopy and fungal cell-membrane potential measurements, although the killing mechanism is not fully understood. MUC7 12-mer possesses antifungal activity in LYM (modified, low salt RPMI 1640 medium) and also exhibits synergistic antifungal effects in vitro with Histatin5 12-mer (Hsn5 12-mer) or miconazole (5).
Information on the antimicrobial activity of the CAMPs in vivo is very limited. To initiate in vivo investigation of the potential of the MUC7 and other antimicrobial peptides as therapeutic agents for use against oral candidiasis, one needs to determine whether or not the peptides retain their activity in saliva. Saliva is a natural ecological environment of oral cavity and some components in saliva may affect therapeutic use of the peptides in vivo. In particular, it is suggested that peptides administered in vivo may be degraded by certain proteases present in saliva, leading to diminishing or loss of their antimicrobial activity. These proteases may come from the host or microorganism in the oral cavity. It has been demonstrated in vitro that the antimicrobial peptides can be degraded by proteases, resulting in decrease or loss of their antimicrobial biological activity (3, 6-8). It is well know that the degradation of proteins or peptides can be prevented by protease inhibitors. It has been suggested that degradation can be also decreased or prevented by modifying molecular structure of peptides by substitution of the natural L-amino acids with D-forms because natural proteases recognize only the natural L-amino acids. Therefore, studies have been conducted aimed at introducing D-amino acids into CAMPs. However, such studies indicate that introduction of D-amino acids into cationic antimicrobial peptides can have either a positive and negative effects on the activity. For example, introduction of three D-amino acids into Magainin-II produced a diastereomer (peptide containing both L- and D-amino acids) with no antimicrobial (antiprotozoan) activity (9). A lack of activity was also observed with other diastereomers (10). On the other hand, all D amino acid Magainin-II exhibited antibacterial potency nearly identical to that of the all-L-enantiomer and was highly resistant to proteolysis and non-hemolytic (11). Similarly, all D-amino acid 11 residue peptide derived from human granulysin (residues 32-42) was resistant to proteolysis and retained the bacteriocidal activity of the L-peptide (12). All D-amino acid isomer of Hsn5 12-mer (known as P113-D) was as active against C. albicans as the natural L-form. In addition, the peptide was active in the presence of sputum from cystic fibrosis patients against respiratory bacteria, while the activity of the L-form was basically lost (13). These studies emphasize the uncertain outcome of using D-isomers and also underline the need for development of new effective antimicrobial agents that are both active and resistant to proteolysis.